Note: Descriptions are shown in the official language in which they were submitted.
WO 99/45957 PCT/EP99/01406
COMBINED VACCINE COMPOSITIONS
This invention relates to novel vaccine formulations, methods for preparing
them
and their use in therapy. In particular the present invention relates to
combination
vaccines for administration to adolescents.
HSV-2 is the primary etiological agent of herpes genitalis. HSV-2 and HSV-1
(the
causative agent of herpes labialis) are characterised by their ability to
induce both
acute diseases and to establish a latent infection, primarily in neuronal
ganglia cells.
Genital herpes is estimated to occur in about 5 million people in the U.S.A.
alone
with 500,000 clinical cases recorded every year (primary and recurrent
infection).
Primary infection typically occurs after puberty and is characterised by the
localised
appearance of painful skin lesions, which persist for a period of between 2 to
3
weeks. Within the following six months after primary infection 50% of patients
will experience a recurrence of the disease. About 25 % of patients may
experience
between 10-15 recurrent episodes of the disease each year. In
immunocompromised
patients the incidence of high frequency recurrence is statistically higher
than in the
normal patient population.
Both HSV-1 and HSV-2 virus have a number of glycoprotein components located
on the surface of the virus. These are known as gB, gC, gD and gE etc.
Vaccines for the prophylaxis of hepatitis B infections, comprising one or more
hepatitis B antigens, are well known. For example the vaccine Engerix-B (Trade
Mark) from SmithKline Beecham Biologicals is used to prevent Hepatitis B. This
vaccine comprises hepatitis B surface antigen (specifically the 226 amino acid
S-
antigen described in Harford et. al. in Postgraduate Medical Journal, 1987, 63
(Suppl. 2), p65-70) and is formulated using aluminium hydroxide as adjuvant.
There is a need for effective combination vaccines to prevent diseases to
which
adolescents are particutarly prone.
CA 02324289 2000-09-07
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01-?3-2060 li4513'? E P 009901406
~4'O-A-92 11291 dcsGribes ap immw~ogtniC hybrid polypeptid~e comprisi~ a !
first
polypcptide component whych is I3tpati~s B surface autigcn covaleatly linked
via a
native sulphul~ atom in the first polypopride component m a second
polypeptic3~;
component such as gD of HSV'.
lA
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WO 99/45957 2 PCT/EP99/01406
The present invention provides a vaccine composition comprising:
(a) a hepatitis B viral (HBV) antigen; and
(b) a herpes simplex viral (HSV) antigen
in combination with an adjuvant which is a preferential stimulator of TH1 cell
response.
The vaccine composition of the invention is of great benefit for
administration to
adolescents who may be particularly at risk of HBV, andlor HSV infection.
Optionally the vaccine composition of the invention additionally comprises one
or
more of a number of other antigens as described below.
It has been found that the vaccine compositions according to the invention
surprisingly show no interference, that is to say that the immune response to
each
antigen in the composition of the invention is essentially the same as that
which is
obtained by each antigen given individually in conjunction with an adjuvant
which
is a preferential stimulator of TH1 cell response.
The vaccine Havrix (Trade Mark), also from SmithKline Beecham Biologicals is
an
example of a vaccine that can be used to prevent hepatitis A infections. It is
_.formulated.with aluminium hydroxide as adjuvant. This vaccine comprises an
attenuated strain of the HM-175 Hepatitis A virus inactivated with formol
(formaldehyde); see Andre et. al. (Frog. med. Virol., vol. 37, pl-24).
As used herein, the term hepatitis A viral (HAV) antigen is used to refer to
either a
protein derived from hepatitis A virus or an attenuated strain of HAV,
optionally
inactivated, e.g. with formaldehyde. If the HAV antigen is a protein derived
from
hepatitis A virus it may optionally be a recombinant protein.
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' The vaccine Twinrix (Trade lVlark) is~a combination of a recombinant
hepatitis B
aaitgen with the aforea~zaroned inactivated attenuated hepatitis A virus. 'fhe
~raccinc may lx used to protect against hepatitis A and hepatitis B
simultaneously.
European gateat EP B- t? 339 667 (Cheruo Sara) descn'bes the general concept
of
combining a hepatitis A antigen and a hepatitis B antigen to make a
eombinatiotr
vaccine. Tn that spcciflcation it is stated that the adjuvant which is used is
riot
critical: it nwst only be capable of enhancing the immune activity to a
desired ex~~t
and not cause any side-effects. It is stated that aluminium gel may be used,
in
1Q paracola~r ahiminiurn hydroxide gel and aluminium phosphate gel.
In a further aspect, the invention provides a vaccine composition coan~isizlg:
(a) a hepatitis 8 viral (HBV) antigen;
(b) a herpes simplex viral (F~,S'V~ antigen; and
(c) an hepatitis A viral (HAV1 antigen
in combination with an ad3uvant which is a preferential stim111atot of Tfi1
cell
response.
5lich a vaccine is of great bcnc~lt for administration to adolescents who may
be
particularly at risk of HBV, andlor I~S'V' irifectiott, andlor HAV infection.
An immune rcspa~sc may be broadly distinguished into two extreme catagorics,
bring a humoral or cell mediated imtuuua responses (traditionally
characterised b;~
2~ antibody and cellular effector mechanisms of protection respectively).
These
categories of response have been termed TFi 1-type responses (cell-mediated
response), and TH2-type ixontutar responses (hutnoral response).
F.,xrreroe TH1-type icnmuue responses may be characterised by the generation
of
antigen specific, haplotype restricted cytotoxic T lymphocytes, and natural
~Iler
cell responses. Ia mice TH1-type responses are often characterised by the
generation of ant~adies of the IgG2a subtype, whilst in tht Intmari thrse
correspond
3
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WO 99/45957 4 PCT/EP99/01406
to IgGI type antibodies. TH2-type immune responses are characterised by the
generation of a broad range of immunoglobulin isotypes including in mice IgGI,
IgA, and IgM.
It can be considered that the driving force behind the development of these
two
types of immune responses are cytokines. High levels of TH1-type cytokines
tend
to favour the induction of cell mediated immune responses to the given
antigen,
whilst high levels of TH2-type cytokines tend to favour the induction of
humoral
immune responses to the antigen.
The distinction of TH1 and TH2-type immune responses is not absolute. In
reality
an individual will support an immune response which is described as being
predominantly TH1 or predominantly TH2. However, it is often convenient to
consider the families of cytokines in terms of that described in murine CD4
+ve T
cell clones by Mosmann and Coffman (Mosmann, T.R. and CofJman, R.L. (1989)
THI and TH2 cells: different patterns of lymphokine secretion lead to
different
functional properties. Annual Review of Immunology, 7, p145-173).
Traditionally,
THl-type responses are associated with the production of the INF-Y and IL-2
cytokines by T-lymphocytes. Other cytokines often directly associated with the
induction of TH1-type immune responses are not produced by T-cells, such as IL-
12. In contrast, TH2- type responses are associated with the secretion of IL-
4, IL-5,
IL-6, IL-10 and tumour necrosis factor-p(TNF-(3).
It is known that certain vaccine adjuvants are particularly suited to the
stimulation
of either TH1 or TH2 - type cytokine responses. Traditionally the best
indicators of
the TH1:TH2 balance of the immune response after a vaccination or infection
includes direct measurement of the production of THI or TH2 cytokines by T
lymphocytes in vitro after restimulation with antigen, and/or the measurement
of the
IgGI:IgG2a ratio of antigen specific antibody responses.
Thus, a TH1-type adjuvant is one which stimulates isolated T-cell populations
to
produce high levels of TH1-type cytokines when re-stimulated with antigen in
vitro,
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- and induces a~gen specific imttauloglobulin responses associated with THi-
type
isatype.
Adjuvants which are capable of preferential stimulation of the TH 1 cell
response arc
descr'bed in Iatcrnational Patent Application No. 'WO 94100153 and WO
9511720~~.
3 De-0-acyla~Ged taonaphosphoryl lipid A (3D-MFL) is one such adjuvant. This
i;~
known from G8 222211 (Ribi). Chemically it is a mixture of 3 De-O-acylated
moaophosphoryl lipid A with 4, 5 or 6 acylated chains arid is a~uta~urad by
Ri'~i
~nu~chem, Montana. A preferred form of 3 Da-t7-acylated monaphosphoryl
lipid A is disclosed in Europaan Patent EP-B- 0 689 454 (SmithKline Beecham
l~iologicals $A).
Preferably, the particles of 3D-Mf'r, are small enough to be sterile filtcnd
througa
a 0.22micron membra~ (as described in L~ropean Patent number 0 689 454).
3D-MPL, will be present is the range of 10~g -100~.g preferably 2S-50~.g per
dose
wherein cho antigen will typically be present in a range 2-SOUg per dose.
Another preferred adjavant comprises QS21, axe Hplc purifud non toxic fraction
derived from the bark of Quillaja Saponaria Molina. OptionaLty this may be
admixed with 3 De-Q-acylatcd monophosphoryl Iipid A (3D-MPL), optionally
together with an carrier'.
E
The method of production of QS21 is disclosed is US patent No: 5,057,540.
Non reactogenic adjuvant formulations containing QS2I harre been described
previously (WO 9b133739). Such formulations comprising QS21 and cholesterol
have been shown to be successful THl stirnulati~g adjuvants when fornmlated
together ~rith as antigen. Thus vaccine compositions which forra part of the
pres~:nt
invention may include a eozubinatioa of QS21 and cholesterol.
5
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23~". ..._ .. _
01=03-2000 EP 009901406
~34513Z
Fltrther sdjuvanu which are preferential stimulators of 'I H"Hi cell response
iacludo
immunomodulatory oligonucleotides, for example unnoethylaied CpG sequences as
disclosed in CVO 96f0~55.
Combinations of different THl stimulating adjuwants, such as otiose wentioned
hereinabove, are also collated as providing an adjuvant which is a
preferetnial
stituulator of TH1 ceh response. For example, Q521 can be formulated together
with 3D-MPL. The ratio of QS2i : 3D-MPL will typically be in the order of 1 :
ZO
to 10 : 1; preferably 1:5 to 5 : I and often substaaiially 1 : 1. 1'he
preferred
I0 range for optimal synergy is 2.5 : 1 to 1 : 13D-MPL: QS21.
Preferably a carxi~er is also present in the vaccine composition according to
the
invention. The corner may be an oil is water emulsion, or alt aht,nainiurn
salt, such
ss aluminium phosphate or aluminium hydroxide.
A preferred oil-in-water emulsion comprises a metabolisible oil, such as
s~u$lone,
alpha tocopherol arid Twecn 80 (Trade aiaxk). Additionally the oil is water
emulsion may contain span 85 audlor lecithin andJor tricaprylin.
In a particutarly preferred aspect the antigens in the vaccine composition
according
to the invention are combined with 3D-MPL. and alum.
Typically for human administration QS21 and 3D-lVipL will be present in a
vaccine
in tire rariae of lpg - 200~,g, such as 10-100pg, preferably l0wg - SO~g per
dose.
Typically the oil in water will comgrisa from 2 to 1 Q ~ sdualene, from 2 to
10 %
alpha tocopherol and from 0.3 to 3 aPo tweea 80. Preferably the ratio of
square:
algba tocopherol is equal to or less than 1 as this provides a atom stable
eraulsion
Span 85 may also be presern at a Ievel of 196. Iri sorae cases it may be
advantageous that the vaccines of the present invention will further contain a
stabiliser.
6
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WO 99/45957 ~ PCT/EP99/01406
Non-toxic oil in water emulsions preferably contain a non-toxic oil, e.g.
squalane or
squalene, an emulsifier, e.g. Tween 80, in an aqueous carrier. The aqueous
carrier
tray be, for example, phosphate buffered saline.
A particularly potent adjuvant formulation involving QS21, 3D-MPL ate
tocopherol in an oil in water emulsion is described in WO 95/17210.
The HSV antigen in the composition of the invention is preferably derived from
HSV-2, typically glycoprotein D. Glycoprotein D is located on the viral
membrane,
and is also found in the cytoplasm of infected cells (Eisenberg R.J. et aI; J
of Virol
1980, 35, 428-435). It comprises 393 amino acids including a signal peptide
and
has a molecular weight of approximately 60 kD. Of all the HSV envelope
glycoproteins this is probably the best characterised (Cohen et al; J. of
Virology,
60, 157-166). In vivo it is known to play a central role in viral attachment
to cell
IS membranes. Moreover, glycoprotein D has been shown to be able to elicit
neutralising antibodies in vivo (Sing et al J. Med. Virology I27: 59-65).
However,
latent HSV-2 virus can still be reactivated and induce recurrence of the
disease
despite the presence of high neutralising antibodies titre in the patients
sera.
An embodiment of the invention is a truncated HSV-2 glycoprotein D of 308
amino
acids which comprises amino acids 1 through 306 naturally occurring
glycoprotein
with the addition Asparagine and Glutamine at the C terminal end of the
truncated
__ . . ... _ _protein_dexoid_.of its membrane anchor region. This-form of the
protein-includes
the signal peptide which is cleaved to yield a mature 283 amino acid protein.
The
production of such a protein in Chinese Hamster ovary cells has been described
in
Genentech's European patent EP-B-139 417.
The recombinant mature HSV-2 glycoprotein D truncate is preferably used in the
vaccine formulations of the present invention and is designated rgD2t.
A combination of this antigen in combination with the adjuvant 3D-MPL has been
described in WO 92/16231.
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WO 99/45957 8 PCT/EP99/01406
The hepatitis B viral (HBV)antigen in the composition of the invention is
typically
hepatitis B surface antigen.
The preparation of Hepatitis B surface antigen (HBsAg) is well documented. See
for example, Harford et. al. in Develop. Biol. Standard 54, page 125 (1983),
Gregg
et. al. in Biotechnology, 5, page 479 (1987), EP-A- 0 226 846, EP-A-0 299 108
and references therein.
As used herein the expression 'Hepatitis B surface antigen', abbreviated
herein to
'HBsAg' or 'HBS' includes any HBsAg antigen or fragment thereof displaying the
antigenicity of HBV surface antigen. It will be understood that in addition to
the
226 amino acid sequence of the HBsAg S antigen (see Tiollais et. al. Nature,
317,
489 (1985) and references therein) HBsAg as herein described may, if desired,
contain all or part of a pre-S sequence as described in the above references
and in
EP-A- 0 278 940. HBsAg as herein described can also refer to variants, for
example the 'escape mutant' described in WO 91/14703. In a further aspect the
HBsAg may comprise a protein described as L* in European Patent Application
Number 0 414 374, that is to say a protein, the amino acid sequence of which
consists of parts of the amino acid sequence of the hepatitis B virus large
(L)
protein (ad or ay subtype), characterised in that the amino acid sequence of
the
protein consists of either:
__.__. _ _. _ _ ._. ..... _ . (a) . . . residues 12 - 52, followed by residues
.133-.T .14~, .followed by _. _ _ _ ._....___.__ ......
residues 175 - 400 of the said L protein; or
(b) residue 12, followed by residues 14 - 52, followed by residues 133 -
145, followed by residues 175 - 400 of the said L protein.
HBsAg may also refer to polypeptides described in EP 0 198 474 or EP 0
304 578.
Normally the HBsAg will be in particle form. It may comprise S protein
alone or may be as composite partic:es, for example (L*,S) wherein L* is as
defined above and S denotes the S-protein of hepatitis B surface antigen.
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~~ 132
The HBsAg may be adsorbed on aluminium phosphate as described in
WG93!?,~ 148.
Preferably the hepatitis 8 (~B'Sl') antigen used in the formulation of the
invention is
I~BsAg S-antigen as used in the commercial product Engcrix-B (Trade Mark;
SynittiRline Beechani Biologicais)_
A vaccine comprising hepatitis B surface antigen in conjunction with 3D-MPL
wa;;
described in European Patent Application EF-A- 0 633 784.
14
Epstein lBarr Virus (EBV), a arember of the herpcsvirus group, causes
infectious
mononucleosis as a primary disease is humans. Predominantly it affects
children
or young adults. More than 90~ of the average adult population is infected by
E~'V that persists fur lifetime in peripheral &lymghocytcs. The virus is
lifelong
produced in the parotid gland arbd spread primarily by exchange of saliva from
individuals who sired the virus. Children infected with EBV arc largely
asymptomatic or have very mild symptoms, while adolescents and adults who
becoiao i~fectcd develop typical izrfectious mononucleosis, characterised by
fever,
pharyngitis, and adenopathy. People who have been infected maintain anti EBV
2U antibodies for the remainder of their lives, and are thus immune to further
infection.
In addition to its infectious qualities, EBV has been sown to transform
lymphocytes into rapidly dividing cells and has therefore been implicated in
several
different lymphomas,including African Burkitt's lymphoma (BL). F.BV may also
be involved in causing nasopbaryngeal carcinoma {NPC). Worldwide it is
estiDaatecl that 80, 000 cases of t~sopb,arynge~a.l carcinoma occur and it is
more
prevalent in ethnic Chinese populations. Infectious mwnonncieosis is a
cansequenc~e
of primary infection by EBY. It is not a life-threarening disease if
additional risk
factors are absent.
Four proteins of the ~,$'V viral envelope constituting the so-called, mtmbrane
az>tsgen complex have been described, They are usually referred to as gp
2201350
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WO 99/45957 ~ ~ PCT/EP99/01406
or gp 2501350 or simply as gp 250 or 350 (see EP-A-151079). There is
convincing
evidence that gp 350 and gp 250 induce the production of neutralising
antibodies
and that antibodies against gp 350 and gp 250 have neutralising capacity.
These
proteins are thus candidates for a possible EBV vaccine. For further
information
about the application of gp 2501350 for prophylaxis and treatment of EBV-
related
diseases see EP 0 173 254.
The major EBV surface glycoprotein gp350/220 infects human target cells
through
interaction with the cellular membrane protein , CD21. Gp350/220 is the
primary
target for EBV-neutralising antibodies in humans and some forms of gp350/220
have been shown to protect against EBV-related disease. Preferably a vaccine
composition according to the invention comprises gp 350 of EBV although other
protective antigens may be used.
Papillomaviruses are small DNA tumour viruses, which are highly species
specific.
As yet, over 70 individual human papillomavirus (HPV) genotypes have been
described. HPVs are generally specific either for the skin (e.g. HPV-1 and -2)
or
mucosal surfaces (e.g. HPV-6 and -11) and usually cause benign tumours (warts)
that persist for several months or years. Such benign tumours may be
distressing
for the individuals concerned but tend not to be life threatening, with a few
exceptions.
... . ... _ ~S,ome HPVs .are also associated with cancers:-~he-strongest
positive association- _ _ . . . ... ...
between an HPV and human cancer is that which exists between HPV-16 and HPV-
18 and cervical carcinoma. Cervical cancer is the most common malignancy in
developing countries, with about 500,000 new cases occurring in the world each
year. It is now technically feasible to actively combat primary HPV-16
infections,
and even established HPV-16-containing cancers, using vaccines. For a review
on
the prospects for prophylactic and therapeutic vaccination against HPV-16 see
Cason J., Clin. Immunother. 1994; 1(4) 293-306 and Hagenesee M.E., Infections
in
Medicine 1997 14(7) 555-556,559-564. Preferably a vaccine composition
according
to the invention comprises the major capsid protein, the L1 protein.
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WO 99/45957 ~ ~ PGT/EP99/01406
Today, the different types of HPVs have been isolated and characterised with
the
help of cloning systems in bacteria and more recently by PCR amplification.
The
molecular organisation of the HPV genomes has been defined on a comparative
basis with that of the well characterised bovine papillomavirus type 1 (BPV1).
Although minor variations do occur, all HPVs genomes described have at least
seven early genes, E1 to E7 and two late genes Ll and L2. In addition, an
upstream regulatory region harbors the regulatory sequences which appears to
control most transcriptional events of the HPV genome.
E1 and E2 genes are involved in viral replication and transcriptional control,
respectively and tend to be disrupted by viral integration. E6 and E7, and
recent
evidence implicate also ES are involved in viral transformation.
In the HPVs involved in cervical carcinoma such as HPV 16 and 18, the
oncogenic
process starts after integration of viral DNA. The integration results in the
inactivation of genes coding for the capsid proteins Ll and L2 and in
installing
continuously over expression of the two early proteins E6 and E7 that will
lead to
gradually loss of the normal cellular differentiation and the development of
the
carcinoma.
Carcinoma of the cervix is common in women and develops through a pre-
- . _ _ .. . _... cancerous .intermediate stage to the invasive care-inoma
which -frequently ieads-to
death. The intermediate stages of the disease is known as cervical
intraepithelial
neoplasia and is graded I to III in terms of increasing severity.
Clinically, HPV infection of the female anogenital tract manifests as cervical
flat
condylomas, the hallmark of which is the koilocytosis affecting predominantly
the
superficial and intermediate cells of the cervical squamous epithelium.
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Koilocytes which are the consequence of a cytopathic effect of the virus,
appear as
multinucleated cells with a perinuclear clear halo. The epithelium is
thickened with
abnormal keratinisation responsible for the warty appearance of the lesion.
Such flat condylomas when positive for the HPV 16 or 18 serotypes, are high-
risk
factors for the evolution toward cervical intraepithelial neoplasia (CIN} and
carcinoma in situ (CIS) which are themselves regarded as precursor lesions of
invasive cervix carcinoma.
International Patent Application No. WO 96/ 19496 discloses variants of human
papilloma virus E6 and E7 proteins, particularly fusion proteins of E6/E7 with
a
deletion in both the E6 and E7 proteins. These deletion fusion proteins are
said to
be immunogenic.
HPV L1 based vaccines are disclosed in W094/00152, W094/20137, W093/02184
and W094/05792. Such a vaccine can comprise the Ll antigen as a monomer, a
capsomer or a virus like particle. Such particles may additionally comprise L2
proteins. Other HPV vaccines are based on the Early proteins, such as E7 or
fusion
proteins such as L2-E7.
In the vaccine of the invention it is preferred to utilise compositions
comprising
either an E6 or E7 protein linked to an immunological fusion partner having T
cell
epitopes. _ . . . ...._. .. ... . . ._...._. _. ....._. ._ __
In a preferred form of the invention, the immunological fusion partner is
derived
from protein D of Heamophilus influenza B. Preferably the protein D derivative
comprises approximately the first 1/3 of the protein, in particular
approximately the
first N-terminal 100-110 amino acids.
Accordingly the present invention in one embodiment comprises antigen (s)
derived
from HPV as described above. Preferably the invention comprises fusion
proteins
comprising Protein D - E6 from HPV I6, Protein D - E7 from HPV 16 Protein D -
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- E7 from HPV 1$ aned Protein D - E6 from HPV 18. xhe protein D part
preferabl.~
co~riprises the first 113 of protein D.
The proteins of the present invention preferably are expressed in E. coli. Ia
a
preferred embodiment the proteins are expressed with a Histidine tail
comprising
between 5 to 9 and preferably six l3isdditle residues. These are advantageous
in
aiding purification. The description of the nlanufaetnre of such proteins is
fbllp
described in co-pending IJh patent applica~ion number G$ 97'17953.5.
In a preferred aspect the vaccine composition of the invention additionally
comprises a Varicclla zostcr viral antigen (VZY antigen). Suitable antigcna of
V'~'Y for inclusion is the vacci,~ formation include gpI-V described by
Longnccker et al., Proc Natl Aced Sci USA 84, 4303-430? (198'!).
In a preferred ernbodianeat gpI saes EIIis et al., US patent 4,y69,239) is
used. See'
also European Patent No. EP-H- 0 405 8~7.
In another preferred aspect tl~ vaccine composition of the invention
additionally
comprises a human cytomegalovirus (HCMV) antigen. HCM'V is a hunnan DL~A
virus belonging to the family of horpes viruses. HCMV is endemic in most parts
of
the ~crrorld. Aonong two populations, HCM'V' is responsible for serious
medical
conditions. HCIIr~T is a ma,~or cause of co~enital defects in new horns. The
second population at risk era immunocompromised patients such as those
sufferia~,
frarn HI'V infection and those patie~s undergoing transplantatiams. The
clinical
~5 disease causes a variety of sympwms including fever, hepatitis, pnaumonitis
and
infectious mononucleosis. A preferred antigen far use in s varxine against
HClvn'
is gH685** as described in WU 95131555. Immunogens for use in HCMV vaccinJs
are also provided by pp65, an HCMV Matrix ~'rotein as descried in W4 94I001~0
(City of Hope).
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WO 99/45957 ~ 4 PCT/EP99/01406
In one preferred aspect the vaccine composition of the invention additionally
comprises both a VZV and an HCMV antigen, in particular those antigens
described above.
In another preferred aspect the vaccine composition of the invention
additionally
comprises a Toxoplasma gondii antigen. Toxoplasma gondii is an obligate
intracellular protozoan parasite responsible for toxoplasmosis in warm-blooded
animals, including man. Although it is generally clinically asymptomatic in
healthy
individuals, toxoplasmosis may cause severe complications in pregnant women
and
immunocompromised patients. A preferred antigen for use in a vaccine against
toxoplasma gondii is SAGl ( also known as P30) as described in W096/02654 or
Tg34 as described in W092/11366.
In one preferred aspect the vaccine composition of the invention additionally
comprises either a VZV antigen or an HCMV antigen combined with a Toxoplasma
gondii antigen, in particular those antigens described above.
In a preferred aspect the vaccine composition of the invention is a
multivalent
vaccine, for example a tetra- or pentavalent vaccine.
The formulations of the present invention are very effective in inducing
protective
immunity, even with very low doses of antigen (e.g. as low as 5pg rgD2t).
They provide excellent protection against primary infection and stimulate,
advantageously both specific humoral (neutralising antibodies) and also
effector cell
mediated (DTH) immune responses.
The present invention in a further aspect provides a vaccine formulation as
herein
described for use in medical therapy, particularly for use in the treatment or
prophylaxis of Herpes Simplex infections and hepatitis B viral infections.
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The vaccine of the present invention will contain an immunoprotective quantity
of
the antigens and may be prepared by conventional techniques.
Vaccine preparation is generally described in Pharmaceutical Biotechnology,
Vo1.61 Vaccine Design - the subunit and adjuvant approach, edited by Powell
and
Newman, Plenurn Press, 1995. New Trends and Developments in Vaccines, edited
by Voller et al., University Park Press, Baltimore, Maryland, U.S.A. 1978.
Encapsulation within liposomes is described, for example, by Fullerton, U.S.
Patent
4,235,877. Conjugation of proteins to macromolecules is disclosed, for
example,
by Likhiie, U.S. Patent 4,372,945 and by Armor et al., U.S. Patent 4,474,757.
The amount of protein in each vaccine dose is selected as an amount which
induces
an immunoprotective response without significant, adverse side effects in
typical
vaccinees. Such amount will vary depending upon which specific immunogen is
employed. Generally, it is expected that each dose will comprise 1-1000pg of
protein, preferably 2-100pg, most preferably 4-40p,g. An optimal amount for a
particular vaccine can be ascertained by standard studies involving
observation of
antibody titres and other responses in subjects. Following an initial
vaccination,
subjects may receive a boost in about 4 weeks.
In addition to vaccination of persons susceptible to HSV or HBV virai
infections,
the pharmaceutical compositions of the present invention may be used to treat,
. ., . __ __. ~ununotherapeuticaily, patients suffering from-the-said viral
infections.
In a further aspect of the present invention there is provided a method of
manufacture as herein described, wherein the method comprises mixing a herpes
viral antigen and a hepatitis B viral antigen with a TH-1 inducing adjuvant,
for
example 3D-MPL and, preferably, a carrier, for example alum.
If desired, other antigens may be added, in any convenient order, to provide
multivalent vaccine compositions as described herein.
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Example 1: Immunogenicity study with ~D + IiBs combination
The objective of the study was to demonstrate the feasibility of the HSV
gD/HBV HBs combination in Al(OH), / 3D-MPL formulation. Immune responses
induced in guinea pigs by immunisation with these antigens used alone or in
combination were compared. HBs is an abbreviation for Hepatitis B surface
antigen, specifically the S-protein as described hereinabove. gD is an
abbreviation
for rgD2t as described hereinabove.
Experimental protocol
The experimental protocol was the following. Groups of 6 female Hartley
guinea pigs were injected intramuscularly on day 0 and day 28 with the
following
formulations:
- group 1: HBs 5 pg / AI(OH)3 125 ~,g / 3D-MPL 12.5 pg
- group 2: gD 5 ~cg / Al(OH) ~ 125 ~,g / 3D-MPL 12.5 ~cg
- group 3: HBs 5 ~cg + gD 5 ~cg / AI(OH) 3 125 p,g / 3D-MPL 12.5 ~cg
- group 4: HBs 5 ~cg + gD 5 ~cg / Al(OH), 125 ~cg / 3D-MPL* 12.5 ~,g
* different 3D-MPL batch
Animals were bled 14 and 31 days after the second immunization. The
humoral immune response against HSV gD and HBV HBs was evaluated at both
time points in ELISA.
Delayed type hypersensitivity (DTH) reactions were also evaluated for HBs.
They consisted in the intradermal injection of 10 p,g HBs in duplicate. The
development of DTH reactions was monitored by measuring skin thickness at 0,
24
and 48 hours after injection.
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17
Results
1. Antibody responses
Anti-gD ELISA titers are shown in Fig. 1. Anti-gD titers in the group
immunized
with gD were comparable to those induced in animals immunized with the gD +
HBs combination. The presence of HBs in the formulation did not affect the
induction of anti-gD antibody responses.
Similarly, anti-HBs antibody titers were compared in animals immunized with
HBs
alone or combined with gD. Fig. 2 shows that comparable anti-HBs titers were
observed in animals immunized with HBs alone or with HBs + gD.
The results are shown in Figures 1-4. from which it may be concluded that in
the
tested formulation, the gD / HBs combination induces antibody responses
comparable to those induced by the same antigens used alone and DTH responses
to
HBs comparable to those induced by HBs alone. Thus no significant differences
in
DTH responses to HBs are observed in HBs or HBs + gD vaccinated animals. The
presence of gD did not affect the DTH response to HBs.
Example 2: PR030 Experiment HBV/HSV combination
The objective of this study was to evaluate in the HSV guinea pig model the
protective efficacy of an HSV gD + HBV HBs combination in a 3D-MPL/alum
formulation as compared to gD alone in a 3D-MPL/alum formulation. The 3D-
MPL/alum formulation comprises alum (10 parts by weight) to 3D-MPL (1 pan by
weight).
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Experimental Protocol
Groups of twelve females Hartley guinea pigs were immunized with the following
formulations or left untreated:
gD + HBs / 3D- gD Al(OH)3, 3D-MPL
MPL/alum (S~eg) (62.S~cg) (6.25~cg)
formulation
+ HBs + + 3D-MPL
(S/cg) Al(OH)3, (6.25~cg)
(62.Spg)
gD / 3D-MPL/alum gD Al(OH)3, 3D-MPL
formulation (S~,g) (125pg) (12.5/cg)
AnitnaIs were immunized intramuscularly twice at days 0 and 28. They were
intravaginally challenged at day 57 (1 month after the second immunization)
with
105 pfu HSV2 MS strain (100,1) and then daily monitored for clinical signs of
primary (days 4 to 12 post-infection) and recurrent (days 13 to 39 post-
infection)
disease. The protection induced was measured according to several criteria
described in Table 1, as well as by cumulative score curves.
Sera collected at days 14 and 28 post II immunization were also tested for
their
anti-gD ELISA Ab titers (expressed as EU/ml); sera obtained at day 28 post II
were also tested for their HSV neutralizing activity ('NEUTRA'; titers
correspond
to the reciprocal of the serum dilution giving 100% protection against HSV2
cytopathic effect).
Results
Serology results:
Immunogenicity data are presented in the following Table and Figure 5:
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Neutra / ELISA ratio
at days 28 post
II
FORMULATIONS ELISA (GMT) NEUTRA (GMT) Ratio NEUTRA
ELISA
gD+HBs / 3D-MPL/alum6687 238 3.5%
formulation
gD ! 3D-MPL/alum 7475 200 2.7
formulation
Similar ELISA and neutralizing titers were induced by the gD (S~cg) + HBs
(S~.g)
combination in 3D-MPL/alum formulation and gD / 3D-MPL/alum formulation.
Protection against primary disease~
As shown in Figure 6 (cumulative score curve) and in Table 1 below, gD + HBs /
3D-MPL/alum formulation combination conferred as good protection against
primary disease as gD alone in 3D-MPL/alum formulation.
Protection against recurrent disease~
- _ . _.._ " r - , Protection against herpes -recurrences . is shown _in
Figure 7 (cumulative - score --
curves) and in Table 2 below. gD + HBs / 3D-MPL/alum formulation combination
conferred as good protection against recurrent disease as gD alone in 3D-
MPL/alum
formulation.
Similar number of animals had recurrences in the gD + HBs and gD alone groups.
Exactly the same number of animals in these groups had more than 1 recurrence
during the observation period (day 13 to 39 post challenge). In those animals
with
recurrences, comparable lesion severity were recorded.
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WO 99/45957 2~ PCT/EP99/01406
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WO 99/45957 2 ~ PCT/EP99/01406
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CA 02324289 2000-09-07 SUBSTITUTE SHEET (RULE 26)
WO 99/45957 22 PCT/EP99/01406
Example 3:
The objective of the study is to evaluate the serological immune responses
induced
in mice by a combination vaccine comprising HAV, HBs and gD formulated with
Aluminium salts and 3D-MPL. The hepatitis A component used in this example
(and abbreviated herein to 'HAV') was the inactivated HMI75 strain found in
Havrix.
Materials and Methods
Antigen / 3D-MPL batches:
HBs: A1:4550 ~cg/ml, pre-adsorbed HBs: 227,62 ~,g/ml
HAV: A1:1380 ~cg/ml, HAV:25230 EU/ml
gD: 493~cg/ml
3D-MPL: 957 ~,g/m1
Formulation process
~ Group I: HBs A1P0, /3D-MPL
HBs I HZO I NaCI / phenoxy I A1P0, for 15 min + 3D-MPL for 1 hr
~ Group 2: gD AlOH3/3D-MPL
H20 / AlOH3 for 5 min + gD for 15 min + 3D-MPL for 30 min + PBS for 15
min + Phenoxy
~ Group3: HAV AIOH,/3D-MPL
H20 /NaCI I Phenoxy for 5 min + AlOH3/HAV for 30 min I 3D-MPL
~ Group 4:
1. HBO I NaCI / Phenoxy for 5 min + A1P0, / HBs for 5 min + 3D-MPL 30 min
2. gD / AlOH3 for 15 min + 3D-MPL for 30 min
Mix 1 and 2 for 20min + HAV for lhr.
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Serolosical read-outs
~ Quantitation of anti-HBs and anti-gD antibody was performed by Elisa using
HBs or gD as coating antigen. Antigen and antibody solutions were used at SO
~cl
per well. Antigen was diluted at a final concentration of 1 ~cg/ml in PBS and
was
adsorbed overnight at 4°c to the wells of 96 wells microtiter plates
(Maxisorb
Immuno-plate, Nunc, Denmark). The plates were then incubated for lhr at
37°c
with PBS containing 1 % bovine serum albumin and 0. I % Tween 20 (saturation
buffer). Two-fold dilutions of sera in the saturation buffer were added to the
antigen-coated plates and incubated for 1 hr 30 min at 37°c. The plates
were
washed four times with PBS 0.1 % Tween 20 and biotin-conjugated anti-mouse Ig
(Amersham, UK) diluted 1 / 1000 in saturation buffer was added to each well
and
incubated for 1 hr 30 min at 37°c. After a washing step, streptavidin-
biotinylated
peroxydase complex (Amersham, UK) diluted 1/5000 or 1/1000 in saturation
buffer (for HBs and anti-gD ELISA respectively) was added for an additional 30
min at 37°c. Plates were washed as above and incubated for 20 min with
a
solution of o-phenylenediamine (Sigma) 0.04 % HZOZ 0.03 % in 0.1 % tween 20
O.OSM citrate buffer pH4.5. The reaction was stopped with HMSO, 2N and read
at 492/620 nm. ELISA titers were calculated from a reference by SofimaxPro
(using a four parameters equation ) and expressed in EU/ml.
~ Quantitation of anti-HAV antibody was performed by Enzymun ELISA
(Boehringer) according to Manufacturer's protocol.
Experimental Protocol
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Groups of 7 BaIb/C mice were immunised intramuscularly with the following
formulations (corresponding to 1/10 human dose):
1. HBs (2 dug) l A1P04 (50 ~cg) / 3D-MPL (5 ~,g)
2. gD (2~cg) / Al(OH)3 (50 ~cg) / 3D-MPL (5 ~cg)
3. HAV (72EU) / Al(OH)3 (50 ~,g) / 3D-MPL (5 ~cg)
4. HAV (72U) / Al(OH)3 (S~cg) + HBs (2 ~.g) l A1P04 (40~cg) / 3D-MPL (2.5 ~cg)
+ gD (2~cg) / Al(OH)3 (5 ~,g) / 3D-MPL(2.5 ~,g)
Animals were immunised twice at day 0 and 21 with SO~d vaccine. Sera were
collected at various time points post-immunisations (21 post I and 14 post II)
and
were tested for their anti-HAV, HBs and gD antibody titers .
Results
Individual data from 21 post I and 14 post II are shown in Table 3 and
summarised
below:
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Table 3:
Anti HBs Anti-HAV Anti-gD
response
response response
(EU/ml) (mIU/ml) (EU/ml)
14 post14 14 post 14 14 post14
I post I post I post
II
II II
Gr.l DHB56A2:
HBs 20 /P04
/ 3D-MPL 50
659 127282
935 165796
979 118574
934 55063
1606 102040
879 90388
409 39447
GMT 880 75015
Gr.2 gD 20
/ OH / 3D-MPL
50
989 149616
1150 97672
564 61866
1087 100172
805 73340
1135 186113
598 122633
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Table 3 coot.
GMT 871 106123
Gr.3 HAV 720 / OH / 3D-MPL
20 20
20 20
20 41
20 20
20 37
20 20
20 20
-
GMT 20 25
Gr.4 HAV 720 / OH HBs P04 - gD
- 20 / 20 /
/ 3D-MPL
25
OH / 3D-MPL 25
88 63427 25 272 202 61711
336 72765 20 28 1166 105676
395 78781 20 498 730 65277
474 99881 20 380 819 62107
890 11243920 20 395 76378
542 12065220 20 493 54908
1020 41460 20 20 345 35466
GMT 414 ~ 79699 21 91 549 69312
~
5 ~ HBs sernlnov
Group Anti-HBs ELISA titers
(EUlml)
Post I Post II
HBs AIP04 3D-MPL 880 75015
HAV Al(OH)3 / HBs A1P04 3D-MPL 414 79699
/ gD
Al(OH)3 3D-MPL
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27
~ gD serology
Group Anti-gD ELISA titers (EU/ml)
Post I Post II
gD Al(OH)3 3D-MPL 871 106123
HAV Al(OH)3 / HBs A1P04 3D-MPL 549 69312
I gD
Al(OH)3 3D-MPL
~ HAV serology
Group Anti-HAV ELISA titers
Post I Post II
HAV Al(OH)3 3D-MPL 20 25
i HAV Al(OH)3 / HBs A1P04 3D-MPL / gD 2I 91
Al(OH)3 3D-MPL
Conclusions
~ Comparable anti-HBs antibody titers in the combination vaccine and in HBs
vaccine containing Aluminium salts and 3D-MPL were observed.
~ Comparable anti-gD antibody titers in the combination vaccine and in gD
vaccine containing Aluminium salts and 3D-MPL were observed.
~ Comparable anti-HAV antibody titers in the combination vaccine and in HAV
vaccine containing Aluminium salts and 3D-MPL were observed.
Thus there appears to be no interference when HBs, gD and HAV are combined in
a vaccine containing aluminium salts and 3D-MPL.
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